Blood/Plasma Distribution Ratio

　　1. Preparation

　　1.1 Use DMSO to prepare the compound into a stock solution with a concentration of 1mM and set aside;

　　1.2 According to the requirements of the experiment, take out fresh whole blood (EDTA-K2 anticoagulant) of different species. Divide the whole blood into 2 parts, centrifuge one part in a centrifuge at 4°C and 4000 rpm for 10 minutes, take the supernatant and obtain plasma;

　　1.3 Prepare a 90% ACN solution (ACN:pure water=9:1);

　　1.4 First, preheat the prepared whole blood and plasma in a water bath at 37°C for 15 minutes;

　　2. Experimental procedures

　　2.1 Use 90% ACN to dilute the 1mM stock into a 100μM working solution;

　　2.2 Use preheated whole blood and plasma to dilute the 100 μM working solution to the final required concentration of 1 μM (according to the experimental arrangement, usually 10 μL working solution + 990 μL matrix);

　　2.3 Take 50 μL of different matrices from different species (make 3 parallel samples) in a 96-well plate, and then add 400 μL of internal standard acetonitrile for protein precipitation, which is used as T0 in the experiment;

　　2.4 Place the prepared sample in a water bath at 37°C and incubate it for 30 minutes;

　　2.5 Centrifuge the whole blood sample in a centrifuge at 37°C and 1750 rpm for 15 minutes to separate the plasma sample;

　　2.6 Add the separated plasma sample and the plasma sample incubated for 30 minutes to a 96-well plate, 50 μL per well, prepare 3 parallel samples for each sample, and then add 400 μL of internal standard acetonitrile to perform protein precipitation on the sample;

　　2.7 Centrifuge in a centrifuge at 4°C at 4000 rpm/min for 20 minutes, take the supernatant, and add formic acid water to reduce the volatilization of acetonitrile (supernatant: formic acid water = 1:1);

　　2.8 Inject samples in LC MS/MS.

　　3. Experimental data calculation

　　3.1 B/P=CB/CP

　　3.2 CB: The concentration of the drug in whole blood (ratio of analyte peak area/internal standard peak area). The whole blood value should be the concentration of the drug directly added to the plasma to represent the whole blood concentration value.

　　3.3 CP: The concentration of the drug in plasma (ratio of analyte peak area/internal standard peak area). The plasma concentration should be the concentration value of the plasma sample after adding the drug to whole blood and centrifuging it.